CARBAPENEMASES FOR USE WITH ANTIBIOTICS FOR THE PROTECTION OF THE INTESTINAL MICROBIOME
This invention relates, in part, to various compositions and methods for protecting the gastrointestinal microbiome from antibiotic disruption.
1 . A method of protecting a subject's gastrointestinal microbiome, comprising administering an effective amount of a pharmaceutical composition comprising a broad spectrum carbapenemase to a subject in need thereof, wherein:
the subject is undergoing treatment or has recently undergone treatment with a carbapenem antibiotic, and
the broad spectrum carbapenemase is capable of degrading or inactivating the carbapenem in the subject's gastrointestinal tract.
2 . (canceled)
3 . The method of claim 1 , wherein the broad spectrum carbapenemase is a metallo-β-lactamase.
4 .- 6 . (canceled)
7 . The method of claim 3 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 95% sequence similarity with SEQ ID NO: [37] 68.
8 . The method of claim 7 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 99% sequence similarity with SEQ ID NO: 68.
9 . (canceled)
10 . The method of claim 1 , wherein the protection of the subject's microbiome comprises treatment or prevention of a microbiome-mediated disorder.
11 . (canceled)
12 . The method of claim [ 1 ] 10 , wherein the microbiome-mediated disorder is one or more of an antibiotic-induced adverse effect, C. difficile infection (CDI), and a C. difficile -associated disease.
13 . The method of claim 12 , wherein the antibiotic-induced adverse effect and/or CDI or C. difficile -associated disease is one or more of: antibiotic-associated diarrhea, C. difficile diarrhea (CDD), C. difficile intestinal inflammatory disease, colitis, and pseudomembranous colitis.
14 . The method of claim 1 , wherein the protection of the subject's microbiome comprises maintenance of a normal intestinal microbiota.
15 . The method of claim 14 , wherein the method treats and/or prevents the overgrowth of one or more pathogenic microorganisms in the GI tract of a subject.
16 . The method of claim 15 , wherein the method treats or prevents a nosocomial infection and/or a secondary emergent infection.
17 . The method of claim 1 , wherein the broad spectrum carbapenemase does not substantially interfere with blood or plasma levels of the antibiotic.
18 . The method of claim 1 , wherein the broad spectrum carbapenemase degrades or inactivates excess or residual antibiotic in the subject's gastrointestinal tract.
19 - 21 . (canceled)
22 . The method of claim 1 , wherein the carbapenem is selected from meropenem, imipenem, ertapenem, and doripenem.
23 - 60 . (canceled)
61 . A method for producing an antibiotic-degrading agent in Escherichia coli ( E. coli ), comprising:
(a) providing a host E. coli cell transformed with a vector comprising a sequence encoding the antibiotic-degrading agent;
(b) culturing the E. coli cell to induce expression of the antibiotic-degrading agent; and
(c) recovering the antibiotic-degrading agent from a soluble fraction prepared from the E. coli cell,
wherein:
the antibiotic-degrading agent is broad spectrum carbapenemase having at least 95% sequence identity with the amino acid sequences of SEQ ID NO: 68 or SEQ ID NO: 53 and
the culturing is in the presence of an amount of zinc sufficient to substantially increase the amount of antibiotic-degrading agent protein in a soluble fraction and reduce the amount of antibiotic-degrading agent protein in inclusion bodies relative to culturing in the absence of zinc.
62 - 76 . (canceled)
77 . The method of claim 61 , wherein the broad spectrum carbapenemase has an amino acid sequence of SEQ ID NO: 68.
78 . The method of claim 61 , wherein the broad spectrum carbapenemase has an amino acid sequence of SEQ ID NO: 53.
79 . The method of claim 8 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 99% sequence similarity with SEQ ID NO: 68 and lacks a leader sequence.
79 . The method of claim 79 , wherein the broad spectrum carbapenemase consists essentially of an amino acid sequence of SEQ ID NO: 68.
80 . The method of claim 1 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 95% sequence similarity with one of SEQ ID NOs: 53 and 54.
81 . The method of claim 1 , wherein the broad spectrum carbapenemase has an amino acid sequence having at least 95% sequence similarity with SEQ ID NO: 38.